Nuclear Localizing Sequences Promote Nuclear Translocation and Enhance the Radiotoxicity of the Anti-CD33 Monoclonal Antibody HuM195 Labeled with 111In in Human Myeloid Leukemia Cells

Journal of Nuclear Medicine

Ross Cameron¹  Mark Minden¹  Judy Wang¹  Kristin Hope¹  John Dick¹  Paul Chen¹  Joseph Brandwein¹  Liqing Jin¹  Raymond M. Reilly¹ 

关键词: acute myelogenous leukemia;    ¹¹¹In;    nuclear localization sequences;    simian virus 40 large T antigen;    Auger electrons;   

DOI  :  

学科分类：医学（综合）

来源: Society of Nuclear Medicine

【 摘 要 】

Our objective was to evaluate the toxicity of the anti-CD33 monoclonal antibody HuM195 modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localizing sequence (NLS; underlined) of simian virus 40 large T antigen and labeled with 111In against acute myeloid leukemia (AML) cells. Methods: HuM195 was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) to introduce maleimide groups for reaction with NLS-peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with 111In. The immunoreactivity of NLS-HuM195 was evaluated by its ability to displace the binding of 111In-HuM195 to HL-60 leukemia cells. Nuclear localization was measured in HL-60 cells by subcellular fractionation. The antiproliferative effects of 111In-NLS-HuM195 and 111In-HuM195 on HL-60, U937, or K562 cells with high, intermediate, or minimal CD33 expression, respectively, were studied. The survival of HL-60 cells or patient AML specimens treated with 111In-NLS-HuM195 or 111In-HuM195 was studied. Normal tissue toxicity was evaluated in BALB/c mice injected intravenously with of 3.7 MBq (22 μg) of 111In-NLS-HuM195 or 111In-HuM195. Results: NLS-HuM195 exhibited relatively preserved CD33 binding affinity (dissociation constant [Kd] = 4.3 ± 1.7 × 10−9 mol/L to 6.9 ± 1.3 × 10−9 mol/L). Nuclear uptake increased from 10.5% ± 0.5% for 111In-HuM195 to 28.5% ± 4.1% or 65.9% ± 1.5% for 111In-HuM195 substituted with 4 or 8 NLS-peptides, respectively. The inhibitory concentrations of 50% (IC50) and 90% (IC90) for HL-60 cells treated with 111In-NLS-HuM195 were 37 kBq per 103 cells and 77–81 kBq per 103 cells, respectively. The IC50 and IC90 values for 111In-HuM195 were 92 kBq per 103 cells and 203 kBq per 103 cells. Growth inhibition was correlated with the level of CD33 expression. The survival of HL-60 cells was reduced from 232 ± 22 colonies (control) to 7 ± 1 colonies with 1.48 mBq per cell of 111In-NLS-HuM195; no colonies were found at 3.33 mBq per cell. The surviving fraction decreased >2-fold in 7 of 9 AML specimens treated with an excess of 111In-NLS-HuM195 and >10-fold in 2 of these specimens. There were no decreases in body weight or hematologic parameters or increases in alanine aminotransferase or creatinine in mice administered 3.7 MBq (22 μg) of 111In-NLS-HuM195 or 111In-HuM195. There was no morphologic damage to the liver or kidneys. Conclusion: We conclude that NLS-peptides routed 111In-HuM195 to the nucleus of AML cells, where the emitted Auger electrons were lethal. 111In-NLS-HuM195 is a promising targeted radiotherapeutic agent for AML.

【 授权许可】

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